L-glutamine Alleviates Osteoarthritis By Up-regulating LncRNA-NKILA Expression Through The TGF-β1/SMAD2/3 Signalling Pathway

Background: OA mainly involves the destruction of the cartilage tissue and the whole joint,such as subchondral sclerosis,osteophyte formation,synovitis,and the infrapatellar fat pad,to name a few.Meanwhile,as a heterogeneous disease with a wide range of underlying pathways,OA is affected by age,body weight,inflammation,trauma,heredity,and other pathogenic factors,each pathogenic factor mainly affects different molecular pathways.L-glutamine(L-Gln)is the most abundant and versatile conditionally essential amino acid in the body(i.e.,it needs to increase the level of amino acid under some conditions,such as pressure).It plays a crucial role in cell composition,nitrogen exchange between organs,homeostasis,regulation of p H,and other physiological metabolic processes.However,the effect L-Gln on OA and its specific molecular mechanism are still unclear.Therefore,the aim of this study was to investigate the therapeutic effects and possible molecular mechanisms of L-Gln on chondrocyte inflammation,rat OA and patients with early knee OA(K-L Ⅰ～Ⅱ).Methods: Chondrocytes were extracted from the knee surface of healthy adult S-D rats and cultured with rat chondrocyte complete culture medium.The effects of L-Gln(0,5,10 and 20 m M)on primary chondrocyte viability were detected by CCK-8 method.IL-1β was used to construct OA model in vitro.The experiment was divided into 5groups: Control group,IL-1β group、IL-1β+5m M L-Gln group,IL-1β+10m M L-Gln group and IL-1β+20m M L-Gln group.Nitrite leveals was detected by Griess reagent.PGE-2 and TNF-α levels were determined by ELISA.WB,IF,IHC,H-E staining,to name a few were used to detect the expression levels in rat’s cartilage of Collagen Ⅱ,Aggrecan,i NOS,COX-2 and MMP-13 in vitro and in vivo,and L-Gln inhibits NF-κB over-activation in rat OA chondrocytes by regulating NKILA induced by the TGF-β1/SMAD2/3 pathway.Ultimately,early knee joint patients were screened by K-L grading,WOMAC scale,VSA and Lequesne score were used to evaluate the knee osteoarthritis after oral administration of L-Gln for 4 weeks.Results: In vitro,L-Gln can upregulate the expression of the lnc RNA-NKILA,which is regulated by the transforming growth factor-β1/SMAD2/3 pathway,and inhibits the activity of nuclear factor-κB,thereby decreasing the expression of i NOS,COX-2,and MMP-13.This leads to a reduction in the generation of NO,PGE-2,TNF-α,and the degradation of the extracellular matrix(i.e.,aggrecan and collagen-Ⅱ)in rat OA chondrocytes.Moreover,intragastric administration of L-Gln reduced the degradation of cartilage tissue and the expression of MMP-13 in a rat OA model.L-Gln also relieved the clinical symptoms of some patients with early knee joint OA.Conclusions: This study reported the anti-inflammatory and protective effects of L-Gln on OA,providing potential evidence for L-Gln as a DMOAD to delay the occurrence and development of OA for future clinical applications.